Cytokine therapy. Cytokines: general information Cytokines and their characteristics

Pro-inflammatory cytokines are synthesized, secreted, and act through their receptors on target cells at an early stage of inflammation, participating in the triggering of a specific immune response, as well as in its effector phase. Below we provide a brief description of the main pro-inflammatory cytokines.

IL-1 - a compound secreted during antigenic stimulation by monocytes, macrophages, Langerhans cells, dendritic cells, keratinocytes, cerebral astrocytes and microglia, endothelial, epithelial, mesothelial cells, fibroblasts, NK-lymphocytes, neutrophils, B-cell lymphocytes, C lymphocytes et al. Approximately 10% of basophils and mast cells also produce IL-1. The listed facts indicate that IL-1 can be secreted directly into the blood, tissue fluid and lymph. All cells in which this cytokine is formed are not capable of spontaneous IL-1 synthesis and respond with its production and secretion in response to the action of infectious and inflammatory agents, microbial toxins, various cytokines, active complement fragments, some active blood coagulation factors, and others. In the figurative expression of A. Bellau, IL-1 is a family of molecules for all occasions. IL-1 is subdivided into 2 fractions, a and b, which are the products of different genes, but have similar biological properties. Both of these forms are formed from the corresponding precursor molecules with the same molecular weight - 31 kDa. As a result of biochemical transformations, single-chain biologically active polypeptides with a molecular weight of 17.5 kDa are ultimately formed. Almost all of the IL-1a remains inside the cell or binds to the membrane. Unlike IL-1a, IL-1b is actively secreted by cells and is the main secretory form of IL-1 in humans. At the same time, both interleukins have the same spectrum of biological activity and compete for binding to the same receptor. However, it should be borne in mind that IL-1a is mainly a mediator of local defense reactions, while IL-1b acts both at the local and systemic levels. Experiments with recombinant IL-1 have shown that this cytokine has no less than 50 different functions, and the targets are cells of almost all organs and tissues. The influence of IL-1 is mainly directed to Th1, although it is able to stimulate Th2 and B-lymphocytes. In the bone marrow, under its influence, the number of hematopoietic cells in the stage of mitosis increases. IL-1 can act on neutrophils, enhancing their locomotor activity and thus promoting phagocytosis. This cytokine is involved in the regulation of the functions of the endothelium and the blood coagulation system, inducing procoagulant activity, the synthesis of proinflammatory cytokines and the expression on the surface of the endothelium of adhesive molecules that provide rolling and attachment of neutrophils and lymphocytes, as a result of which leukopenia and neutropenia develop in the vascular bed. Acting on liver cells, it stimulates the formation of acute phase proteins. It has been established that IL-1 is the main mediator of the development of local inflammation and acute phase response at the level of the organism. In addition, it accelerates the growth of blood vessels after damage. Under the influence of IL-1 in the blood, the concentration of iron and zinc decreases and the excretion of sodium increases. Finally, it has recently been found that IL-1 is capable of increasing the amount of circulating nitric oxide. The latter is known to play an extremely important role in the regulation of blood pressure, promotes platelet disaggregation and enhances fibrinolysis. It should be noted that under the influence of IL-1 the formation of rosettes of neutrophils and lymphocytes with platelets is enhanced, which plays an important role in the implementation of nonspecific resistance, immunity and hemostasis (Yu.A. Vitkovsky). All this suggests that IL-1 stimulates the development of a whole complex of protective reactions of the body, aimed at limiting the spread of infection, eliminating invading microorganisms and restoring the integrity of damaged tissues. IL-1 has an effect on chondrocytes, osteoclasts, fibroblasts and pancreatic b-cells. Under its influence, the secretion of insulin, ACTH and cortisol increases. The addition of IL-1b or TNFa to the primary culture of pituitary cells decreases the secretion of thyroid-stimulating hormone.

IL-1 is produced in the central nervous system, where it can act as a transmitter. Under the influence of IL-1, sleep occurs, accompanied by the presence of a-rhythm (slow wave sleep). It also promotes the synthesis and secretion of nerve growth factor by astrocytes. It has been shown that the content of IL-1 increases during muscle work. Under the influence of IL-1, the production of IL-1 itself, as well as IL-2, IL-4, IL-6, IL-8 and TNFa, is enhanced. The latter, in addition, induces the synthesis of IL-1, IL-6 and IL-8.

Many pro-inflammatory effects of IL-1 are carried out in combination with TNFa and IL-6: fever induction, anorexia, influence on hematopoiesis, participation in nonspecific anti-infectious protection, secretion of acute phase proteins, and others (A.S. Simbirtsev).

IL-6- a monomer with a molecular weight of 19-34 kDa. It is produced by stimulated monocytes, macrophages, endotheliocytes, Th2, fibroblasts, hepatocytes, Sertoli cells, cells of the nervous system, thyrocytes, cells of the islets of Langerhans, etc. Together with IL-4 and IL-10, it provides the growth and differentiation of B-lymphocytes, facilitating the transition the latter in antibodies. In addition, it, like IL-1, stimulates hepatocytes, leading to the formation of acute phase proteins. IL-6 acts on hematopoietic progenitor cells and, in particular, stimulates megakaryocytopoiesis. This compound has antiviral activity. There are cytokines belonging to the IL-6 family - oncostatin M (OnM), a factor that inhibits leukemia, ciliary neurotropic factor, cardiotropin-1. Their influence does not affect the immune system. The IL-6 family has an effect on embryonic stem cells, causes myocardial hypertrophy, CWA synthesis, maintenance of proliferation of myeloma cells and hematopoietic progenitors, differentiation of macrophages, osteoclasts, nerve cells, increased thrombocytopoiesis, etc.

It should be noted that mice with targeted inactivation (knockout) of the gene encoding a common receptor component for the IL-6 family of cytokines develop numerous abnormalities in various body systems that are incompatible with life. Along with the impairment of cardiogenesis in the embryos of such mice, there is a sharp decrease in the number of progenitor cells of various hematopoietic rows, as well as a sharp decrease in the size of the thymus. These facts indicate the extreme importance of IL-6 in the regulation of physiological functions (A.A. Yarilin).

There are very complex mutually regulating relationships between pro-inflammatory cytokines, which act as synergists. Thus, IL-6 inhibits the production of IL-1 and TNFa, although both of these cytokines are inducers of IL-6 synthesis. In addition, IL-6, acting on the hypothalamic-pituitary system, leads to an increase in the production of cortisol, which inhibits the expression of the IL-6 gene, as well as genes of other pro-inflammatory cytokines.

The IL-6 family also includes oncostatin M (OnM), possessing an extremely broad spectrum of action. Its molecular weight is 28 kDa. It has been found that OnM is able to inhibit the growth of a number of tumors. It stimulates the formation of IL-6, a plasminogen activator, vasoactive intestinal peptides, and CWA. It follows from the above that OnM should play an important role in the regulation of the immune response, blood coagulation and fibrinolysis.

IL-8 belongs to the so-called family of chemokines that stimulate chemotaxis and chemokinesis and number up to 60 individual substances with their own structural and biological properties. Mature IL-8 exists in several forms, differing in the length of the polypeptide chain. The formation of one form or another depends on specific proteases acting on the N-terminus of the non-glycosylated precursor molecule. Depending on which cells synthesize IL-8, it contains a different number of amino acids. The greatest biological activity is possessed by the form of IL-8, consisting of 72 amino acids (A.S. Simbirtsev).

IL-8 is released by polymorphonuclear leukocytes, monocytes, macrophages, megakaryocytes, neutrophils, T lymphocytes (Tx), fibroblasts, chondrocytes, keratinocytes, endothelial and epithelial cells, hepatocytes and microglia.

IL-8 is produced in response to the action of biologically active compounds, including proinflammatory cytokines, as well as IL-2, IL-3, IL-5, GM-CSF, various mitogens, lipopolysaccharides, lectins, and viral decay products, while anti-inflammatory cytokines (IL-4, IL-10) reduce the production of IL-8. Its activation and release also occurs under the influence of thrombin, a plasminogen activator, streptokinase and trypsin, which indicates a close relationship between the function of this cytokine and the hemostatic system.

The synthesis of IL-8 is carried out on the action of a wide variety of endogenous or exogenous stimuli that arise in the focus of inflammation during the development of a local defense reaction to the introduction of a pathogenic agent. In this respect, IL-8 production has many similarities with other pro-inflammatory cytokines. At the same time, the synthesis of IL-8 is suppressed by steroid hormones, IL-4, IL-10, Ifa and Ifg.

IL-8 stimulates chemotaxis and chemokinesis of neutrophils, basophils, T-lymphocytes (to a lesser extent) and keratinocytes, causing degranulation of these cells. With intravascular injection of IL-8, there is a rapid and sharp granulocytopenia, followed by an increase in the level of neutrophils in the peripheral blood. In this case, neutrophils migrate to the liver, spleen, lungs, but not to damaged tissues. Moreover, the experiment has shown that intravenous administration of IL-8 blocks the migration of neutrophils into the intradermal areas of inflammation.

In unstimulated neutrophils, IL-8 causes the release of a protein associated with vitamin B 12 from specific granules and gelatinase from secretory vesicles. Degranulation of azurophilic granules in neutrophils occurs only after their stimulation with cytochalasin-B. At the same time, elastase, myeloperoxidase, b-glucoronidase and other elastases are released, and the expression of adhesive molecules on the leukocyte membrane occurs, ensuring the interaction of the neutrophil with the endothelium. It should be noted that IL-8 is not capable of triggering a respiratory burst, but may enhance the effect of other chemokines on this process.

IL-8 is able to stimulate angiogenesis by activating proliferative processes in endotheliocytes and smooth muscle cells, which plays an important role in tissue repair. In addition, it can inhibit IL-4-induced IgE synthesis.

Apparently, IL-8 plays an important role in the local immunity of the mucous membranes. In healthy people, it is found in the secretions of the salivary, lacrimal, sweat glands, in colostrum. It has been found that smooth muscle cells in the human trachea are capable of producing negligible amounts of IL-8. Under the influence of bradykinin, IL-8 production increases 50 times. Protein synthesis blockers inhibit the synthesis of IL-8. There is every reason to believe that locally IL-8 provides the course of protective reactions when exposed to pathogenic flora in the upper respiratory tract.

IL-12 discovered more than ten years ago, but its properties have been studied only in recent years. It is produced by macrophages, monocytes, neutrophils, dendritic cells and activated B-lymphocytes. To a much lesser extent, IL-12 is capable of secreting keratinocytes, Langerhans cells and resting B-lymphocytes. In addition, it is produced by microglial cells and astrocytes, which requires their cooperation. IL-12 is a heterodimer consisting of two covalently linked polypeptide chains: heavy (45 kDa) and light (35 kDa). Biological activity is inherent only in the dimer; each of the individual chains does not possess similar properties.

Nevertheless, the main target cells for IL-12 remain NK, T-lymphocytes (CD4 + and CD8 +) and, to a lesser extent, B-lymphocytes. It can be considered that it serves as a link between macrophages and monocytes, contributing to an increase in the activity of Th1 and cytotoxic cells. Thus, this cytokine makes a significant contribution to the provision of antiviral and antitumor protection. The inducers of IL-12 synthesis are microbial components and pro-inflammatory cytokines.

IL-12 belongs to heparin-binding cytokines, which suggests its participation in the process of hemostasis.

In recent years, it has been shown that IL-12 is a key cytokine for enhancing the cell-mediated immune response and effective anti-infectious protection against viruses, bacteria, fungi and protozoa. The protective effects of IL-12 in infections are mediated by Ifg-dependent mechanisms, enhanced nitric oxide production and T-cell infiltration. However, its main effect is to synthesize Ifg. The latter, while accumulating in the body, promotes the synthesis of IL-12 by macrophages. The most important function of IL-12 is to direct Tx0 differentiation towards Tx1. In this process, IL-12 is a synergist for Ifg. Meanwhile, after differentiation, Th1 ceases to need IL-12 as a co-stimulating molecule. The nature of the immune response largely depends on IL-12: whether it will develop according to cellular or humoral immunity.

One of the most important functions of IL-12 is a sharp increase in the differentiation of B-lymphocytes into antibody-producing cells. This cytokine is used to treat patients with allergies and bronchial asthma.

IL-12 has an inhibitory effect on the production of IL-4 by T-lymphocytes of memory, mediated through APC. In turn, IL-4 suppresses the production and secretion of IL-12.

IL-12 synergists are IL-2 and IL-7, although both of these cytokines often act on different target cells. The physiological antagonist and inhibitor of IL-12 is IL-10, a typical anti-inflammatory cytokine that inhibits Th1 function.

IL-16 - is secreted by T-lymphocytes, mainly stimulated by CD4 +, CD8 +, eosinophils and bronchial epithelial cells. Increased secretion of IL-16 was found when T cells were treated with histamine. By chemical nature, it is a homotetramer with a molecular weight of 56000-80000 D. It is an immunomodulatory and pro-inflammatory cytokine, because it is a chemotactic factor for monocytes and eosinophils, as well as T-lymphocytes (CD4 +), enhancing their adhesion.

It should be noted that pretreatment of CD4 + with recombinant IL-16 suppresses HIV-1 promoter activity by approximately 60%. Based on the above facts, a hypothesis has been put forward, according to which the effect of IL-16 on HIV-1 replication is observed at the level of viral expression.

IL-17 formed by macrophages. At present, recombinant IL-17 has been obtained and its properties have been studied. It turned out that under the influence of IL-17 human macrophages intensively synthesize and secrete pro-inflammatory cytokines - IL-1b and TNFa, which is in direct proportion to the dose of the studied cytokine. The maximum effect is observed approximately 9 hours after the start of incubation of macrophages with recombinant IL-17. In addition, IL-17 stimulates the synthesis and secretion of IL-6, IL-10, IL-12, PgE 2, RIL-1 antagonist, and stromalysine. Anti-inflammatory cytokines - IL-4 and IL-10 - completely cancel the IL-17-induced release of IL-1b, while GTFb 2 and IL-13 only partially block this effect. IL-10 suppresses the induced release of TNFa, while IL-4, IL-13 and GTFb 2 suppress the secretion of this cytokine to a lesser extent. The presented facts strongly suggest that IL-17 should play an important role in the initiation and maintenance of the inflammatory process.

IL-18in terms of biological effects, it is a functional doubler and synergist of IL-12. The main producers of IL-18 are macrophages and monocytes. Its structure is extremely similar to IL-1. IL-18 is synthesized in the form of an inactive precursor molecule, which requires the participation of an IL-1b-converting enzyme to convert it into an active form.

Under the influence of IL-18, the antimicrobial resistance of the body increases. In bacterial infection, IL-18, together with IL-12 or Ifa / b, regulates Ifg production by Tx and NK cells and enhances the expression of Fas ligand on NK and T lymphocytes. Recently, it has been found that IL-18 is a CTL activator. Under its influence, the activity of CD8 + cells in relation to cells of malignant tumors increases.

Like IL-12, IL-18 promotes the preferential differentiation of Th0 into Th1. In addition, IL-18 leads to the formation of GM-CSF and thereby enhances leukopoiesis and inhibits the formation of osteoclasts.

IL-23consists of 2 subunits (p19 and p40) that make up IL-12. Separately, each of the listed subunits does not have biological activity, however, together they, like IL-12, enhance the proliferative activity of T-lymphoblasts and Ifg secretion. IL-23 has a weaker activity than IL-12.

TNFis a polypeptide with a molecular weight of about 17 kDa (consists of 157 amino acids) and is divided into 2 fractions - a and b. Both fractions have approximately the same biological properties and act on the same cellular receptors. TNFa is secreted by monocytes and macrophages, Th1, endothelial and smooth muscle cells, keratinocytes, NK lymphocytes, neutrophils, astrocytes, osteoblasts, etc. To a lesser extent, TNFa is produced by some tumor cells. The main inducer of TNFa synthesis is bacterial lipopolysaccharide, as well as other components of bacterial origin. In addition, the synthesis and secretion of TNFa are stimulated by cytokines: IL-1, IL-2, Ifa and b, GM-CSF, etc. Epstein-Barr virus, Ifa / b, IL-4, IL-6, IL- 10, G-CSF, TGFb, etc.

The main manifestation of the biological activity of TNFa is the effect on some tumor cells. At the same time, TNFa leads to the development of hemorrhagic necrosis and thrombosis of the supplying blood vessels. At the same time, under the influence of TNFa, the natural cytotoxicity of monocytes, macrophages and NK cells increases. The regression of tumor cells occurs especially intensively with the combined action of TNFa and Ifg.

Under the influence of TNFa, the synthesis of lipoprotein kinase is inhibited, one of the main enzymes that regulate lipogenesis.

TNFa, as a mediator of cytotoxicity, is able to inhibit cell proliferation, differentiation, and functional activity of many cells.

TNFa is directly involved in the immune response. It plays an extremely important role in the early moments of the onset of the inflammatory reaction, because it activates the endothelium and promotes the expression of adhesive molecules, which leads to adhesion of granulocytes to the inner surface of the vessel. Under the influence of TNFa, transendothelial migration of leukocytes to the inflammatory focus occurs. This cytokine activates granulocytes, monocytes and lymphocytes and induces the production of other pro-inflammatory cytokines - IL-1, IL-6, Ifg, GM-CSF, which are TNFa synergists.

Formed locally, TNFa in the focus of inflammation or an infectious process sharply increases the phagocytic activity of monocytes and neutrophils and, enhancing the processes of peroxidation, contributes to the development of complete phagocytosis. Together with IL-2, TNFa significantly increases Ifg production by T-lymphocytes.

TNFa is also involved in the processes of destruction and repair, as it causes the growth of fibroblasts and stimulates angiogenesis.

In recent years, it has been established that TNF is an important regulator of hematopoiesis. Directly or in conjunction with other cytokines, TNF affects all types of hematopoietic cells.

Under its influence, the function of the hypothalamus-pituitary-adrenal glands system, as well as of some endocrine glands - the thyroid gland, testicles, ovaries, pancreas and others is enhanced (A.F. Vozianov).

Interferonsare formed by almost any cells of the human body, but mainly their production is carried out by blood and bone marrow cells. The synthesis of interferons occurs under the influence of antigenic stimulation, although a very small concentration of these compounds can be found normally in the bone marrow, bronchi, various organs of the gastrointestinal tract, skin and others. The level of interferon synthesis is always higher in nondividing cells than in rapidly dividing cells.

Cytokines are low molecular weight protein substances that are produced by almost all immune cells. They serve as a kind of chemical mediators within the immune system. But they cannot be called only immune factors, since they take part in the processes of hematopoiesis, intersystem signal transmission and have the ability to interact with cells of other organs and systems, which allows maintaining the constancy of the internal environment. These substances provide control over the reactions of inflammation and hypersensitivity, and under certain conditions contribute to damage to the body's own tissues.

Cytokines are important components of the inflammatory process that are necessary for the implementation of the protective functions of the immune system. Proinflammatory cytokines, growth factors, chemokines are involved in the development of these reactions. However, in some cases, it is necessary to suppress and contain the inflammatory process. For this there are anti-inflammatory cytokines.

General properties

The cytokine binds to a receptor on the cell membrane, which stimulates the cell to perform its function.

All cytokines not only have their own individual characteristics, but also have common functional features:

• To perform their function, they bind to a specific receptor on the cell membrane.
• Some of them interact with various target cells, others - only with certain cell lines.
• The synthesis of these substances is impulsive. They have a fairly short half-life and short duration of action.
• Cytokines are effective at very low concentrations.
• They can cause local reactions or systemic effects.
• Cytokines interact with each other. So, one of them can influence the activity of the other, stimulating, strengthening or weakening it.
• They are characterized by overlapping redundant functions (the same effect is caused by several cytokines).
• The same cell is capable of producing different cytokines.
• One type of cytokine can be produced by different cells.

Pro-inflammatory cytokines

Cytokines with pro-inflammatory activity begin to be secreted in the body as a result of damage or penetration of an infectious agent. They are produced by activated lymphocytes, cells of the monocytic series, dendritic cells, etc. The most important representatives of this group of cytokines are:

• interleukin-1;
• interleukin-6;
• tumor necrosis factor α;
• interleukin-17 and 18.

Cytokines responsible for the inflammatory response are synthesized and secreted into the pathological focus rather quickly. They appear there within an hour and begin to exert their effect, forming a zone of inflammation:

• induce the expression of membrane receptors that are sensitive to inflammatory factors;
• enhance the movement of leukocytes from the bloodstream to the pathological focus;
• stimulate the synthesis of other cytokines with a similar effect;
• cause fever;
• increase the production of protein substances of the acute phase of inflammation;
• activate the activity of the nervous system and endocrine glands.

It should be noted that in high concentrations, these substances are capable of causing pathological reactions. The most prominent example is septic shock.

Interleukin-1 combines about 11 classes of protein molecules. 5 of them are active cytokines, the functions of the rest are unknown. Any cells of the body can be targets for interleukin-1, but the most sensitive to it are:

• vascular endothelium;
• leukocytes;
• chondrocytes;
• epithelial cells;
• nervous tissue.

Under its influence, more than 50 types of biological reactions are realized in the body. It activates all pro-inflammatory genes, causes the migration of leukocyte cells to the inflammatory focus, while increasing their phagocytic activity and bactericidal effect. It also affects vascular tone and blood circulation in this area. In addition, interleukin-1 has multiple systemic effects:

• acts on the hypothalamus and causes a temperature reaction;
• takes part in the development of general manifestations of the inflammatory process (general weakness, weakness, poor appetite, drowsiness);
• enhances;
• stimulates the release of granulocytes from the hematopoietic zone of the bone marrow;
• in case of damage to cartilage and bone tissue, it can cause their destruction, etc.

Interleukin-6 is a broad-spectrum cytokine. It takes part in the induction of almost the entire complex of local inflammatory reactions, but its effect is weaker than interleukin-1 or TNF-α. However, it does not increase the production of other cytokines, but, on the contrary, inhibits it, thus combining the opposite properties of pro- and anti-inflammatory cytokines.

Tumor necrosis factor α is produced in the body mainly by cells of the monocytic-macrophage system. This cytokine has a fairly broad spectrum of activity. It first appears in the blood after induction of inflammation (among all pro-inflammatory cytokines). Its action is similar to the effects of interleukin-1, but more pronounced. It also enhances the expression of adhesion molecules, the synthesis of various inflammatory factors, accelerates the movement of leukocytes and activates them. In addition, it enhances the bacterial potential of phagocytes and stimulates the growth and development of fibroblasts. With an increased local concentration of TNF-α, tissue damage occurs, and with an increase in its concentration in the blood, severe toxic effects develop.

Anti-inflammatory cytokines

Along with the existence of factors that cause an inflammatory response, cytokines are produced in the human body that can suppress it. The relationship between them is an important point in the regulation of the onset and development of inflammation, because not only the course of the pathological process, but also its outcome depends on this. The main representatives of this group of cytokines are:

• interleukin-4;
• interleukin-10;
• interleukin-13;
• transforming growth factor beta.

Interleukin-4 is produced by type 2 T-helpers. It is an antagonist of γ-interferon, inhibits the secretion of TNF-α, interleukin-1, interleukin-6 and inhibits the activity of macrophages and T-lymphocytes. Together with other cytokines, it promotes the proliferation of tissue basophils.

Also, type 2 T-helpers produce interleukin-10 and 13, which reduce the synthesis of cytokines responsible for the development of inflammation, and increase the proliferation of mast cells and B-lymphocytes. As a result, cellular immunity is suppressed and humoral immunity is stimulated (production of antibodies).

Transforming growth factor beta is synthesized by a variety of cell types, including macrophages and lymphocytes. Its main function is considered to suppress the activity and growth of T-lymphocytes, as well as macrophages, neutrophils, natural killer cells. It inhibits the immune response and stimulates the reparative processes in the body by increasing collagen synthesis.

Conclusion

Interleukin 13 is a cytokine that suppresses the inflammatory process.

The role of cytokines in the body is very important. Taking into account their diverse regulatory properties, it becomes clear that insufficient or excessive secretion of these substances is important in various diseases and pathological processes. Currently, drugs are being developed on the basis of cytokines and their receptors, which are used in oncology, transplantology and other branches of medicine.

This chapter will consider an integrated approach to assessing the cytokine system using previously described modern research methods.

First, we outline the basic concepts of the cytokine system.

Cytokines are currently considered as protein-peptide molecules produced by various cells of the body and carrying out intercellular and intersystemic interactions. Cytokines are universal regulators of the life cycle of cells, they control the processes of differentiation, proliferation, functional activation and apoptosis of the latter.

Cytokines produced by cells of the immune system are called immunocytokines; they are a class of soluble peptide mediators of the immune system necessary for its development, functioning and interaction with other systems of the body (Kovalchuk L.V. et al., 1999).

As regulatory molecules, cytokines play an important role in the reactions of innate and adaptive immunity, ensure their interconnection, control hematopoiesis, inflammation, wound healing, the formation of new blood vessels (angiogenesis), and many other vital processes.

Currently, there are several different classifications of cytokines, taking into account their structure, functional activity, origin, type of cytokine receptors. Traditionally, in accordance with biological effects, it is customary to distinguish the following groups of cytokines.

1. Interleukins(IL-1-IL-33) are secretory regulatory proteins of the immune system that provide mediator interactions in the immune system and its connection with other systems of the body. Interleukins are classified according to their functional activity into pro- and anti-inflammatory cytokines, lymphocyte growth factors, regulatory cytokines, etc.

3. Tumor necrosis factors (TNF)- cytokines with cytotoxic and regulatory actions: TNFα and lymphotoxins (LT).

4. Hematopoietic cell growth factors- stem cell growth factor (Kit - ligand), IL-3, IL-7, IL-11, erythropoietin, trobopoietin, granulocyte-macrophage colony-stimulating factor - GM-CSF, granulocyte CSF - G-CSF, macrophage -

ny KSF - M-KSF).

5. Chemokines- С, CC, СХС (IL-8), СХ3С - regulators of chemotaxis of various types of cells.

6. Non-lymphoid cell growth factors- regulators of growth, differentiation and functional activity of cells of various tissue belonging (fibroblast growth factor - FGF, growth factor of endothelial cells, epidermal growth factor - EGF of the epidermis) and transforming growth factors (TGFβ, TGFα).

Among others, in recent years, a factor that inhibits the migration of macrophages (migration inhibiting factor - MIF), which is considered as a neurohormone with cytokine and enzyme activity, has been actively studied (Suslov A.P., 2003; Kovalchuk L.V. et al.,

Cytokines differ in structure, biological activity, and other properties. However, along with the differences, cytokines have general properties,characteristic of this class of bioregulatory molecules.

1. Cytokines are generally glycosylated polypeptides of average molecular weight (less than 30 kD).

2. Cytokines are produced by cells of the immune system and other cells (for example, endothelium, fibroblasts, etc.) in response to an activating stimulus (pathogen-associated molecular structures, antigens, cytokines, etc.) and participate in the reactions of innate and adaptive immunity, regulating their strength and duration. Some cytokines are synthesized constitutively.

3. The secretion of cytokines is a short-term process. Cytokines are not stored as preformed molecules, but their

synthesis always begins with gene transcription. Cells produce cytokines in low concentrations (picograms per milliliter).

4. In most cases, cytokines are produced and act on target cells in the immediate vicinity (short-range action). The main site of action of cytokines is the intercellular synapse.

5. Redundancysystem of cytokines is manifested in the fact that each type of cell is capable of producing several cytokines, and each cytokine can be secreted by different cells.

6. All cytokines are characterized by pleiotropy,or the polyfunctionality of the action. Thus, the manifestation of signs of inflammation is due to the influence of IL-1, TNF-α, IL-6, IL-8. Duplication of functions ensures the reliability of the cytokine system.

7. The action of cytokines on target cells is mediated by highly specific high-affinity membrane receptors, which are transmembrane glycoproteins, usually consisting of more than one subunit. The extracellular part of the receptors is responsible for cytokine binding. There are receptors that eliminate the excess of cytokines in the pathological focus. These are the so-called decoy receptors. Soluble receptors are the extracellular domain of the membrane receptor, separated by an enzyme. Soluble receptors are able to neutralize cytokines, participate in their transport to the inflammation focus and in their excretion from the body.

8. Cytokines work on the principle of a network.They can act in concert. Many functions initially attributed to a single cytokine appear to be mediated by the concerted action of several cytokines (synergismactions). Examples of synergistic interactions of cytokines are stimulation of inflammatory responses (IL-1, IL-6 and TNF-a), as well as IgE synthesis

(IL-4, IL-5 and IL-13).

Some cytokines induce the synthesis of other cytokines (cascade).The cascade action of cytokines is necessary for the development of inflammatory and immune responses. The ability of some cytokines to enhance or weaken the production of others determines important positive and negative regulatory mechanisms.

The antagonistic effect of cytokines is known, for example, the production of IL-6 in response to an increase in the concentration of TNFα can be

a negative regulatory mechanism for controlling the production of this mediator during inflammation.

Cytokine regulation of the functions of target cells is carried out using autocrine, paracrine or endocrine mechanisms. Some cytokines (IL-1, IL-6, TNFα, etc.) are able to participate in the implementation of all of the above mechanisms.

The cell's response to the influence of a cytokine depends on several factors:

From the type of cells and their initial functional activity;

From local cytokine concentration;

From the presence of other mediator molecules.

Thus, producer cells, cytokines and their specific receptors on target cells form a single mediator network. It is the set of regulatory peptides, and not individual cytokines, that determine the final cell response. At present, the cytokine system is considered as a universal system of regulation at the level of the whole organism, which ensures the development of protective reactions (for example, during infection).

In recent years, the idea of \u200b\u200ba cytokine system has developed that combines:

1) producer cells;

2) soluble cytokines and their antagonists;

3) target cells and their receptors (Fig. 7.1).

Violations of various components of the cytokine system lead to the development of numerous pathological processes, and therefore the identification of defects in this regulatory system is important for the correct diagnosis and appointment of adequate therapy.

Let us first consider the main components of the cytokine system.

Cytokine-producing cells

I. Lymphocytes represent the main group of cytokine-producing cells in the adaptive immune response. Resting cells do not secrete cytokines. With antigen recognition and with the participation of receptor interactions (CD28-CD80 / 86 for T-lymphocytes and CD40-CD40L for B-lymphocytes), cell activation occurs, leading to the transcription of cytokine genes, translation and secretion of glycosylated peptides into the intercellular space.

Figure: 7.1.Cytokine system

CD4 T-helpers are represented by subpopulations: Th0, Th1, Th2, Th17, Tfh, which differ in the spectrum of secreted cytokines in response to various antigens.

Th0 produce a wide range of cytokines at very low concentrations.

Direction of differentiation Th0determines the development of two forms of an immune response with a predominance of humoral or cellular mechanisms.

The nature of the antigen, its concentration, localization in the cell, the type of antigen-presenting cells, and a certain set of cytokines regulate the direction of Th0 differentiation.

After antigen capture and processing, dendritic cells present antigenic peptides to Th0 cells and produce cytokines that regulate the direction of their differentiation into effector cells. The role of individual cytokines in this process is shown in Fig. 7.2. IL-12 induces the synthesis of IFNγ by T-lymphocytes and] HPC. IFNu provides differentiation of Th1, which begin to secrete cytokines (IL-2, IFNu, IL-3, TNF-a, lymphotoxins), which regulate the development of reactions to intracellular pathogens

(delayed-type hypersensitivity (HRT) and various types of cellular cytotoxicity).

IL-4 ensures differentiation of Th0 into Th2. Activated Th2 produce cytokines (IL-4, IL-5, IL-6, IL-13, etc.), which determine the proliferation of B-lymphocytes, their further differentiation into plasma cells, and the development of antibody responses, mainly to extracellular pathogens.

IFNu negatively regulates the function of Th2 cells and, conversely, IL-4, IL-10 secreted by Th2 inhibit Th1 function (Fig. 7.3). The molecular mechanism of this regulation is associated with transcription factors. The expression of T-bet and STAT4, determined by IFNy, directs the differentiation of T cells along the Th1 pathway and suppresses the development of Th2. IL-4 induces the expression of GATA-3 and STAT6, which, respectively, ensures the conversion of naive THO into Th2 cells (Fig. 7.2).

In recent years, a special subpopulation of T helper cells (Th17) producing IL-17 has been described. Members of the IL-17 family can be expressed by activated memory cells (CD4CD45RO), u5T cells, NKT cells, neutrophils, monocytes under the influence of IL-23, IL-6, TGFβ, produced by macrophages and dendritic cells. The main differentiating factor in humans is ROR-C, in mice - ROR-γ l The cardinal role of IL-17 in the development of chronic inflammation and autoimmune pathology has been shown (see Fig. 7.2).

In addition, T-lymphocytes in the thymus can differentiate into natural regulator cells (Treg) expressing surface markers CD4 + CD25 + and the transcription factor FOXP3. These cells are able to suppress the immune response mediated by Th1 and Th2 cells through direct cell-cell contact and the synthesis of TGFβ and IL-10.

Diagrams of differentiation of Th0 clones and cytokines secreted by them are shown in Fig. 7.2 and 7.3 (see also color insert).

T-cytotoxic cells (CD8 +), natural killer cells are weak producers of cytokines, such as interferons, TNFα and lymphotoxins.

Excessive activation of one of the Th subpopulations can determine the development of one of the variants of the immune response. Chronic imbalance of Th activation can lead to the formation of immunopathological conditions associated with the manifestation of

allergies, autoimmune pathology, chronic inflammatory processes, etc.

Figure: 7.2.Various subpopulations of T-lymphocytes producing cytokines

II. In the innate immune system, the main cytokine producers are myeloid cells. With the help of Toll-like receptors (TLRs), they recognize similar molecular structures of various pathogens, the so-called pathogen-associated molecular patterns (RAMP), for example, lipopolysaccharide (LPS) of gram-negative bacteria, lipoteichoic acids, peptidoglycans of gram-positive microorganisms, flagellin, DNA rich in G repetitions, etc. As a result

this interaction with TLR triggers an intracellular signal transduction cascade leading to the expression of genes of two main groups of cytokines: proinflammatory and IFN type 1 (Fig. 7.4, see also color inset). Mainly, these cytokines (IL-1, -6, -8, -12, TNFa, GM-CSF, IFN, chemokines, etc.) induce the development of inflammation and are involved in the body's defense against bacterial and viral infections.

Figure: 7.3.The spectrum of cytokines secreted by TH1 and TH2 cells

III. Cells that are not related to the immune system (cells of connective tissue, epithelium, endothelium) constitutively secrete autocrine growth factors (FGF, EGF, TGFR, etc.). and cytokines that support the proliferation of hematopoietic cells.

Cytokines and their antagonistsare described in detail in a number of monographs (Kovalchuk L.V. et al., 2000; Ketlinsky S.A., Simbirtsev A.S.,

Figure: 7.4.TLR-mediated induction of cytokine production by innate immune cells

Overexpression of cytokines is unsafe for the body and can lead to the development of an excessive inflammatory response, an acute phase response. Various inhibitors are involved in the regulation of the production of pro-inflammatory cytokines. Thus, a number of substances have been described that nonspecifically bind the cytokine IL-1 and prevent the manifestation of its biological action (a2-macroglobulin, C3-component of complement, uromodulin). Specific inhibitors of IL-1 include soluble decoy receptors, antibodies, and an IL-1 receptor antagonist (IL-1RA). With the development of inflammation, the expression of the IL-1RA gene increases. But even normally, this antagonist is present in the blood in high concentrations (up to 1 ng / ml or more), blocking the action of endogenous IL-1.

Target cells

The action of cytokines on target cells is mediated through specific receptors that bind cytokines with very high affinity, and individual cytokines can use

common receptor subunits. Each cytokine binds to its specific receptor.

Cytokine receptors are transmembrane proteins and are divided into 5 main types. The most common is the so-called hematopoietin type of receptor, which has two extracellular domains, one of which contains a common sequence of amino acid residues of two repeats of tryptophan and serine, separated by any amino acid (WSXWS motif). The second type of receptor can have two extracellular domains with a large number of conserved cysteines. These are receptors of the IL-10 and IFN family. The third type is represented by cytokine receptors belonging to the TNF group. The fourth type of cytokine receptor belongs to the superfamily of immunoglobulin receptors, which have extracellular domains that are structurally similar to the domains of immunoglobulin molecules. The fifth type of receptor that binds molecules of the chemokine family is represented by transmembrane proteins that cross the cell membrane in 7 places. Cytokine receptors can exist in a soluble form, retaining the ability to bind ligands (Ketlinsky S.A. et al., 2008).

Cytokines can influence proliferation, differentiation, functional activity and apoptosis of target cells (see Fig. 7.1). The manifestation of the biological activity of cytokines in target cells depends on the participation of various intracellular systems in signal transmission from the receptor, which is associated with the characteristics of the target cells. The signal to apoptosis is carried out, among other things, with the help of a specific region of the TNF receptor family, the so-called “death” domain (Fig. 7.5, see color inset). Differentiation and activating signals are transmitted through the intracellular Jak-STAT proteins - signal transducers and activators of transcription (Fig. 7.6, see color inset). G-proteins are involved in signaling from chemokines, which leads to increased migration and cell adhesion.

A comprehensive analysis of the cytokine system includes the following.

I. Evaluation of producer cells.

1. Definition of expression:

Receptors that recognize a pathogen or antigen TCR, TLR) at the level of genes and protein molecules (PCR, flow cytometry);

Adapter molecules that conduct a signal that triggers the transcription of cytokine genes (PCR, etc.);

Figure: 7.5.Signal transmission from the TNF receptor

Figure: 7.6.Jak-STAT - signaling pathway from type 1 cytokine receptors

Cytokine genes (PCR); protein molecules of cytokines (assessment of the cytokine synthesizing function of human mononuclear cells).

2. Quantification of subpopulations of cells containing certain cytokines: Th1, Th2 Th17 (method of intracellular staining of cytokines); determination of the number of cells secreting certain cytokines (ELISPOT method, see Ch. 4).

II. Assessment of cytokines and their antagonists in biological media of the body.

1. Testing the biological activity of cytokines.

2. Quantitative determination of cytokines using ELISA.

3. Immunohistochemical staining of cytokines in tissues.

4. Determination of the ratio of opposing cytokines (pro- and anti-inflammatory), cytokines and cytokine receptor antagonists.

III. Evaluation of target cells.

1. Determination of the expression of cytokine receptors at the level of genes and protein molecules (PCR, flow cytometry).

2. Determination of signaling molecules in the intracellular content.

3. Determination of the functional activity of target cells.

At present, numerous methods have been developed for assessing the cytokine system, which provide diverse information. Among them are distinguished:

1) molecular biological methods;

2) methods for the quantitative determination of cytokines using immunoassay;

3) testing the biological activity of cytokines;

4) intracellular staining of cytokines;

5) ELISPOT method, which allows detecting cytokines around a single cytokine-producing cell;

6) immunofluorescence.

We give a brief description of these methods.

Via molecular biological methodsit is possible to study the expression of genes of cytokines, their receptors, signaling molecules, to study the polymorphism of these genes. In recent years, a large number of studies have been carried out that have revealed associations between variants of alleles of genes of molecules of the cytokine system and

to a number of diseases. The study of allelic variants of cytokine genes can provide information on the genetically programmed production of one or another cytokine. The most sensitive is the real-time polymerase chain reaction - RT-PCR (see Chapter 6). Hybridization method in situallows to clarify the tissue and cellular localization of the expression of cytokine genes.

The quantitative determination of cytokines in biological fluids and in cultures of peripheral blood mononuclear cells by ELISA can be characterized as follows. Since cytokines are local mediators, it is more expedient to measure their levels in the corresponding tissues after extraction of tissue proteins or in natural fluids, for example, in tears, lavage from cavities, urine, amniotic fluid, cerebrospinal fluid, etc. Cytokine levels in serum or other body fluids reflect the current state of the immune system, i.e. synthesis of cytokines by cells of the body in vivo.

Determination of levels of cytokine production by peripheral blood mononuclear cells (MNCs) shows the functional state of cells. Spontaneous production of MNC cytokines in culture indicates that cells are already activated in vivo.The synthesis of cytokines induced (by various stimulants, mitogens) reflects the potential, reserve ability of cells to respond to an antigenic stimulus (in particular, to the action of drugs). Decreased induced production of cytokines can serve as one of the signs of an immunodeficiency state. Cytokines are not specific for a particular antigen. Therefore, a specific diagnosis of infectious, autoimmune and allergic diseases by determining the level of certain cytokines is impossible. At the same time, the assessment of cytokine levels allows obtaining data on the severity of the inflammatory process, its transition to the systemic level and prognosis, the functional activity of cells of the immune system, the ratio of Th1 and Th2 cells, which is very important in the differential diagnosis of a number of infectious and immunopathological processes.

In biological media, cytokines can be quantified using a variety of immunoassay methods,using polyclonal and monoclonal antibodies (see chapter 4). ELISA allows you to find out what are the exact concentrations of cytokines in bio-

logical body fluids. Enzyme-linked immunosorbent assay of cytokines has a number of advantages over other methods (high sensitivity, specificity, independence from the presence of antagonists, the possibility of accurate automated accounting, accounting standardization). However, this method also has its limitations: ELISA does not characterize the biological activity of cytokines, it can give false results due to cross-reacting epitopes.

Biological testingcarried out on the basis of knowledge of the basic properties of cytokines, their action on target cells. The study of the biological effects of cytokines allowed the development of four types of cytokine testing:

1) by the induction of proliferation of target cells;

2) by cytotoxic effect;

3) by induction of differentiation of bone marrow progenitors;

4) by antiviral action.

IL-1 is determined by the stimulating effect on the proliferation of murine thymocytes activated by the mitogen in vitro;IL-2 - by the ability to stimulate the proliferative activity of lymphoblasts; TNFa and lymphotoxins are tested for cytotoxic action on mouse fibroblasts (L929). Colony stimulating factors are evaluated for their ability to support the growth of bone marrow progenitors in the form of colonies in agar. The antiviral activity of IFN is detected by the inhibition of the cytopathic action of viruses in the culture of human diploid fibroblasts and the tumor line of fibroblasts of mice L-929.

Cell lines have been created whose growth depends on the presence of certain cytokines. Table 7.1 is a list of cell lines used for cytokine testing. According to the ability to induce the proliferation of sensitive target cells, biotesting of IL-1, IL-2, IL-4, IL-6, IL-7, IL-15, etc. is carried out. However, these testing methods are not sufficiently sensitive and informative. Molecules of inhibitors and antagonists can mask the biological activity of cytokines. Several cytokines exhibit general biological activity. Nevertheless, these methods are ideal for testing the specific activity of recombinant cytokines.

Table 7.1.Cell Lines Used to Test the Biological Activity of Cytokines

The end of the table. 7.1

Lab 7-1

Determination of the biological activity of IL-1 by the comitogenic effect on the proliferation of mouse thymocytes

The method of biological testing of IL-1 is based on the ability of a cytokine to stimulate the proliferation of murine thymocytes.

IL-1 can be determined in the culture of monocytes stimulated with LPS, as well as in any biological body fluid.It is necessary to pay attention to a number of details.

1. For testing, the thymocytes of C3H / HeJ mice stimulated to proliferation by mitogens (concanavalin A - ConA and phytohemagglutinin - PHA) are used. Thymocytes C3H / HeJ were not chosen by chance: mice of this inbred line do not respond to LPS, which can be present in the test material and cause IL-1 production.

2. Thymocytes respond to IL-2 and mitogens; therefore, the presence of IL-2 and mitogens should also be determined in preparations tested for IL-1.

Operating procedure

1. Receive a suspension of thymocytes at a concentration of 12 × 10 6 / ml of medium RPMI 1640 containing 10% serum of embryos of cows and 2-mercaptoethanol (5 × 10 -5 M).

2. Prepare a series of successive two-fold dilutions of experimental (biological body fluids) and control samples. Biological fluids containing IL-1 or samples obtained during incubation of mononuclear cells without LPS and laboratory standard IL-1-containing preparation are used as controls. In 96-well round bottom plates, 50 μl are transferred from each dilution into 6 wells.

3. In three wells of each dilution add 50 μl of purified PHA (Wellcome) dissolved in complete medium at a concentration of 3 μg / ml, and in the other 3 wells - 50 μl of medium.

4. Add 50 μl of thymocyte suspension to each well and incubate for 48 h at 37 ° C.

6. Before the end of cultivation, 50 μl of a solution (1 μCi / ml) of ["3 H] -thymidine is added to the wells and incubated for another 20 hours.

7. To determine the level of radioactivity, the culture cells are transferred onto filter paper using an automatic cell harvester, the filters are dried and the inclusion of the label is determined by a liquid scintillation counter.

8. Results are expressed as a stimulation factor.

where m cp is the average number of pulses in 3 holes.

If thymocytes respond to stimulation with standard IL-1, then the stimulation index of the test sample exceeding 3 reliably indicates IL-1 activity.

Bioassay is the only method for assessing cytokine function, but this method must be complemented by various types of appropriate control for specificity using monoclonal antibodies. The addition of certain monoclonal antibodies to the cytokine into the culture blocks the biological activity of the cytokine, which proves that the detected cytokine serves as a signal for cell line proliferation.

Using bioassay to detect interferon.The principle of assessing the biological activity of IFN is based on its antiviral effect, which is determined by the degree of inhibition of the multiplication of the test virus in cell culture.

Cells sensitive to the action of IFN can be used in this work: primarily trypsinized fibroblast cells of chicken and human embryos, transplanted cells of human diploid fibroblasts, and mouse cell culture (L929).

When assessing the antiviral action of IFN, it is advisable to use viruses with a short reproduction cycle, high sensitivity to the action of IFN: mouse encephalomyelitis virus, mouse vesicular stomatitis, etc.

Lab 7-2

Determination of interferon activity

1. A suspension of diploid fibroblasts of a human fetus on a medium with 10% serum of bovine embryos (cell concentration - 15-20 × 10 6 / ml) is poured into sterile 96-well flat-bottomed plates, 100 μl per well and placed in a CO 2 incubator at a temperature 37 ° C.

2. After the formation of a complete monolayer, the growth medium is removed from the wells and 100 µl of the support medium is added to each well.

3. Titration of the IFN activity in the samples under study is carried out by the method of two-fold dilutions on a monolayer of fibroblasts.

Simultaneously with the samples, the mouse encephalomyelitis virus (VEM) is introduced into the wells at a dose that causes 100% cell damage 48 hours after infection.

4. For control use wells with intact (untreated) cells infected with the virus.

In each study, reference IFN samples with known activity are used as reference drugs.

5. Plates with sample dilutions are incubated for 24 hours at 37 ° C in an atmosphere with 5% CO 2.

6. The level of IFN activity is determined by the reciprocal of the maximum dilution of the test sample, which inhibits the cytopathic effect of the virus by 50%, and is expressed in units of activity per ml.

7. To determine the type of IFN, antiserum against IFNα, IFNβ or IFNγ is added to the system. The antiserum cancels the action of the corresponding cytokine, which makes it possible to identify the type of IFN.

Determination of the biological activity of the migration of the inhibitory factor.At present, completely new ideas have been formed about the nature and properties of the MYTH, discovered in the 60s of the last century as a mediator of cellular immunity and for many years left without due attention (Bloom B.R., Bennet B., 1966; David J.R., 1966). Only in the last 10-15 years has it become clear: MYTH is one of the most important biological mediators in the body with a wide range of biological functions of cytokine, hormone, enzyme. The action of MIF on target cells is realized through the CD74 - receptor or through the nonclassical pathway of endocytosis.

MYTH is considered as an important mediator of inflammation, activating the function of macrophages (cytokine production, phagocytosis, cytotoxicity, etc.), as well as an endogenous immunoregulatory hormone that modulates glucocorticoid activity.

More and more information is accumulating about the role of MIF in the pathogenesis of many inflammatory diseases, including sepsis, rheumatoid arthritis (RA), glomerulonephritis, etc. In RA, the concentration of MIF in the fluid of the affected joints is significantly increased, which correlates with the severity of the disease. Under the influence of MYTH, the production of pro-inflammatory cytokines by both macrophages and synovial cells increases.

There are various methods for testing the activity of MIF, when migrating cells (target cells for MIF) are placed in a glass capillary (capillary test), in a drop of agarose or in an agarose well.

We present a relatively simple screening method based on the formation of cell microcultures (leukocytes or macrophages), standard in area and number of cells, at the bottom of the wells of a 96-well flat-bottomed plate, followed by their cultivation in a nutrient medium and determining the change in the area of \u200b\u200bthese microcultures under the action of MIF ( Suslov A.P., 1989).

Lab 7-3

Definition of MYTH activity

Determination of the biological activity of MIF is carried out using a device for the formation of cell microcultures (Fig. 7.7) - MIGROSKRIN (Research Institute of Epidemiology and Microbiology named after NF Gamaleya RAMS).

1. In the wells of a 96-well plate (Flow, Great Britain or similar) add 100 μl of a sample diluted in culture medium, in which the MYTH activity is determined (each dilution in 4 parallels, experimental samples). The culture medium contains RPMI 1640, 2 mM L-glutamine, 5% fetal bovine serum, 40 μg / ml gentamicin.

2. Add culture medium (in 4 parallels) to control wells, 100 µl each.

3. Prepare a cell suspension of peritoneal macrophages, for which 2 hybrid mice (CBAxC57B1 / 6) F1 are injected intraperitoneally with 10 ml of Hanks solution with heparin (10 U / ml), gently massage the abdomen for 2-3 minutes. Then the animal is killed by decapitation, the abdominal wall is carefully pierced in the groin area and the exudate is sucked out through the needle with a syringe. The cells of peritoneal exudate are washed twice with Hanks solution, centrifuged for 10-15 minutes at 200 g. Then a cell suspension is prepared with a concentration of 10 ± 1 million / ml of RPMI 1640 medium. Counting is carried out in a Goryaev chamber.

4. Assemble the MIGROSKRIN system, which is a rack for directed and standard fixation of tips with cell cultures in a strictly vertical position at a given height above the center of the well of a 96-well culture plate, and also includes 92 tips for an automatic pipette from Costar, USA (Fig. . 7.7).

Insert the tripod legs into the corner wells of the plate. The cell suspension is drawn up with an automatic pipette into tips - 5 μl each, rinsed from excess cells by a single dip into the medium and inserted vertically into the sockets of the system rack. The filled rack with tips is kept at room temperature for 1 hour on a strictly horizontal surface. During this time, the cells of the suspension settle to the bottom of the wells, where standard cell microcultures are formed.

5. The tip rack is carefully removed from the plate. A plate with a microculture of cells is placed in a strictly horizontal position in a CO 2 incubator, where it is cultured for 20 hours. During cultivation, cells migrate along the bottom of the well.

6. Quantitative recording of results after incubation is carried out on a binocular magnifier, visually assessing the size of the colony on the scale inside the eyepiece. Microcultures are circular. The researchers then determine the average colony diameter from the measurements of the colonies in 4 test or control wells. The measurement error is ± 1 mm.

The migration index (MI) is calculated by the formula:

The sample has MYTH-activity if the MI values \u200b\u200bare equal

For the conventional unit (U) of the MYTH activity, the reciprocal is taken, equal to the value of the highest dilution of the sample (sample), at which the migration index is 0.6 ± 0.2.

Biological activity of FEOα is assessed by its cytotoxic effect on the line of transformed fibroblasts L-929. Recombinant TNFα was used as a positive control, and cells in a culture medium were used as a negative control.

Calculate the cytotoxic index (CI):

where a- the number of living cells in the control; b- the number of living cells in the experiment.

Figure: 7.7.MIGROSKRIN scheme - devices for quantitative assessment of cell culture migration

Cells are stained with a dye (methylene blue), which is incorporated only into dead cells.

The value of the reciprocal dilution of the sample required to obtain 50% of cellular cytotoxicity is taken as a conventional unit of TNF activity. Specific activity of the sample - the ratio of activity in arbitrary units per 1 ml to the concentration of the protein contained in the sample.

Intracellular cytokine staining.A change in the ratio of cells producing various cytokines may reflect the pathogenesis of the disease and serve as a criterion for the prognosis of the disease and for evaluating the therapy.

The intracellular staining method determines the expression of a cytokine at the level of one cell. Flow cytometry allows you to count the number of cells expressing a particular cytokine.

Let us list the main steps in the determination of intracellular cytokines.

Unstimulated cells produce small amounts of cytokines, which, as a rule, are not deposited; therefore, an important stage in the assessment of intracellular cytokines is the stimulation of lymphocytes and blockade of the release of these products from cells.

The most commonly used cytokine inducer is the protein kinase C activator phorbol-12-myristate-13-acetate (PMA) in combination with the calcium ionophore ionomycin (IN). The use of such a combination causes the synthesis of a wide range of cytokines: IFNu, IL-4, IL-2, TNFα. The disadvantage of using PMA-IN is the problem of detecting CD4 molecules on the surface of lymphocytes after such activation. Also, the production of cytokines by T-lymphocytes is induced by mitogens (PHA). B cells and monocytes stimulate

Mononuclear cells are incubated in the presence of inducers of cytokine production and a blocker of their intracellular transport, brefeldin A or monensin, for 2-6 hours.

The cells are then resuspended in a buffer solution. For fixation, 2% formaldehyde is added, incubated for 10-15 minutes at room temperature.

Then the cells are treated with saponin, which increases the permeability of the cell membrane, and stained with monoclonal antibodies specific to the detected cytokines. Pre-staining of surface markers (CD4, CD8) increases the amount of information obtained about the cell and allows you to more accurately determine its population.

There are some limitations in the application of the methods described above. So, with their help, it is impossible to analyze the synthesis of cytokines by a single cell, it is impossible to determine the number of cytokine-producing cells in a subpopulation, it is impossible to determine whether cytokine-producing cells express unique markers, whether different cytokines are synthesized by different cells or by the same ones. The answer to these questions is obtained using other research methods. To determine the frequency of cytokine-producing cells in a population, the method of limiting dilutions and a variant of the ELISPOT enzyme-linked immunosorbent assay are used (see Chapter 4).

In situ hybridization method.The method includes:

2) fixation with paraformaldehyde;

3) detection of mRNA using labeled cDNA. In some cases, cytokine mRNA is determined on sections using radioisotope PCR.

Immunofluorescence.The method includes:

1) freezing the organ and preparing cryostat sections;

2) fixation;

3) processing the sections with fluorescein-labeled anti-cytokine antibodies;

4) visual observation of fluorescence.

These techniques (hybridization in situand immunofluorescence) are fast and do not depend on the threshold concentrations of the secreted product. However, they do not measure the amount of secreted cytokine and can be technically complex. A variety of careful monitoring for non-specific reactions is required.

Using the presented methods for assessing cytokines, pathological processes associated with disturbances in the cytokine system at various levels were identified.

Thus, the assessment of the cytokine system is extremely important for characterizing the state of the body's immune system. The study of various levels of the cytokine system provides information on the functional activity of different types of immunocompetent cells, on the severity of the inflammatory process, on its transition to the systemic level and on the prognosis of the disease.

Questions and tasks

1. List the general properties of cytokines.

2. Give the classification of cytokines.

3. List the main components of the cytokine system.

4. List the cytokine-producing cells.

5. Describe the families of cytokine receptors.

6. What are the mechanisms of functioning of the cytokine network?

7. Tell us about the production of cytokines in the innate immune system.

8. What are the main approaches to a comprehensive assessment of the cytokine system?

9. What are the methods for testing cytokines in biological body fluids?

10. What are the defects in the cytokine system in various pathologies?

11. What are the main methods of biological testing of IL-1, IFN, MIF, TNFa in biological fluids?

12. Describe the process of determining the intracellular content of cytokines.

13. Describe the process of determining the cytokines secreted by a single cell.

14. Describe the sequence of methods used to detect a defect at the cytokine receptor level.

15. Describe the sequence of methods used to detect a defect at the level of cytokine-producing cells.

16. What information can be obtained by examining the production of cytokines in the culture of mononuclear cells, in the blood serum?

mD, prof. Tsaregorodtseva T.M., head. laboratory of immunology

Central Research Institute of Gastroenterology of the Moscow Department of Health

Cytokines (CK) play an important role in the development and course of diseases of various organs and systems, including the digestive system. CK - low molecular weight proteins, endogenous biologically active mediators that provide signal transmission, information exchange between different types of cells within one organ, communication between organs and systems, both under physiological conditions and under the action of various pathogenic factors. In healthy individuals, CKs are produced in minimal amounts sufficient for the manifestation of a biological effect; in pathological conditions, their content increases manifold.

CK are synthesized by activated cells, mainly lymphocytes, monocytes, tissue macrophages. Different cells, for example, macrophages, lymphocytes, endothelial cells, can synthesize the same CK. On the other hand, the same cells can produce different CKs.

CK synthesis is genetically programmed, short-term, and regulated by inhibitors. The increased content of CK can be caused not only by an increase in their synthesis, but also by a violation of catabolism, timely excretion from the body in case of liver and kidney damage.

The increased synthesis of CK leads to the activation of many different types of cells. Thus, a wide interaction is realized at the subcellular, cellular, organ, systemic levels, the formation of a complex defense reaction aimed at neutralizing damaging agents, their destruction, elimination from the body, preservation of its homeostasis, structural and functional integrity.

Cytokine classification

Currently, more than 100 CCs have been identified, and their number continues to grow. Among the CK, the following main groups are distinguished: interleukins (IL), interferons (IF), tumor necrosis factors (TNF), growth factors, chemokines, etc.

Mechanisms of action

CKs realize their biological effect by binding to receptors localized on the membranes of target cells - immunocompetent, endothelial, epithelial, smooth muscle and other specialized cells. Outside the cells, CKs can bind to circulating receptors, which transport them to the lesion focus and remove them from the vascular bed. The synthesis of receptors proceeds more intensively and for a longer time than the synthesis of CK, which contributes to a more complete realization of their biological effect and removal from the body.

Functional properties

CKs have a wide range of biological properties: they induce and regulate physiological and pathological processes such as growth, proliferation, cell differentiation, metabolism, inflammation, and immune response. CCs are multifunctional, universal, pleiotropic. The same CKs can interact with receptors of different cells, while CKs with a similar structure can have different biological effects, and structurally different CKs can cause the same effect.

In the body, CKs closely interact with each other, forming a universal network that launches and regulates a cascade of inflammatory, immune, metabolic processes, both local and systemic, aimed at neutralizing and eliminating pathogenic agents. This communication biological system has a significant margin of safety due to the duplication of most of the functions of different CK, their interchangeability, a combination of autocrine and paracrine regulation. Nevertheless, with all the variety of functions, specific CCs predominate certain properties developed in the process of evolution.

Cytokines and inflammation

Proinflammatory CK (IL-1β, IL-6, IL-8, IL-12, IFN-γ, TNF-α) are characterized by a wide range of biological action on numerous target cells. IL-1β under the action of pathogenic factors is one of the first to be included in the response of the body, activating T- and B-lymphocytes, initiating the synthesis of IL-6, TNF-α, PG, having a pyrogenic effect. IL-6 is produced mainly by lymphocytes, but hepatocytes, Kupffer cells, endothelium, bile duct epithelial cells, and fibroblasts can participate in its synthesis. IL-6 has not only pro, but also anti-inflammatory effect, completes the acute phase of inflammation, activates B-lymphocytes, regulates the proliferation of liver cells, bile ducts, the formation of fibrosis, the formation of granulomas. IL-8 - chemokine - stimulates and regulates the adhesion, chemotaxis of leukocytes to the lesion. TNF-α is a key multifunctional CK of systemic action, plays a dominant role in the development of local and general pathological processes, stimulates the synthesis of proinflammatory ILs, the proliferation of endothelial cells, and regulates the tone of blood vessels. TNF-α enhances oxidative stress, has a powerful cytotoxic effect, and induces necrosis of tumor, infected and other affected cells. By stimulating cytotoxic, phagocytic activity, utilization of defective cells, neutralizing bacterial toxins, TNF-α takes part in the formation of the body's defense reactions. However, intensive prolonged synthesis of this CK promotes hemodynamic disorder, the development of hyperthermia, cachexia, necrosis, toxic septic shock, and multiple organ failure. IL-12, stimulates the synthesis of IFN-γ - a universal immunomodulator that increases the adhesive, cytotoxic, phagocytic activity of cells, has an antiproliferative, antiviral effect.

Anti-inflammatory cytokines - IL-4, -10, -13, -17 - inhibit inflammation, inhibit the synthesis of pro-inflammatory CK, the formation of highly active metabolites of oxygen and nitrogen. IL-4 stimulates the proliferation and differentiation of B-lymphocytes into plasma cells, the synthesis of immunoglobulins, antibodies, and the humoral immune response. This is a brief description of the main biological functions of key CKs regulating both local and systemic inflammatory processes. Inflammation is a universal reaction that develops in the body in response to the action of various damaging factors. Most diseases of the digestive system - gastritis, pancreatitis, hepatitis, cholecystitis and others - are largely due to the development of inflammation. CK regulate the intensity, prevalence and duration of inflammation. On the one hand, proinflammatory CK enhance the phenomena of alteration, destruction, stimulate the synthesis of acute phase proteins, oxidative stress. On the other hand, the early development of adequate inflammatory processes contributes to limiting the lesion focus, increasing barrier functions, regeneration, healing of a tissue defect, and preventing systemic complications.

Cytokines and the immune response

CKs are directly involved in the formation of both nonspecific defense and a specific immune response, forming in the complex a single integrative cellular-humoral system of the body's defense under the action of pathogenic agents. In cases where the damaging factor is a carrier of genetically foreign information, inflammatory processes include immune mechanisms. The main cells that implement the immune response are macrophages, T- and B-lymphocytes, plasma cells. However, many tissue cells (endothelium, epithelium, smooth muscle, liver, etc.) take part in the immune response, interacting with immunocompetent cells. The leading role in the development and regulation of the immune response belongs to T-lymphocytes, the population of which includes T-helpers, T-suppressors, cytotoxic T-lymphocytes. Helper T cells (Tx) produce CKs with different functional properties. Тх 1 type synthesize IFN-γ, IL-2, TNF-α; Type 11 th - IL-4, -5, -6, -10, -13, inducing, respectively, cellular and humoral immune response. In the lamina propria and Peyer's patches of the gastrointestinal tract, Tx type 11 is mainly localized, stimulating the humoral immune response directed against numerous bacterial antigens that affect the gastrointestinal mucosa, and is mainly realized by IgA.

Cytokines play a leading role in the regulation of the main stages of the immune response. Depending on the nature of the pathogenic agent, the intensity, duration of antigenic stimulation, and the initial state of the body's immune system, CK can act as antagonists and synergists, complementing each other. In diseases of the digestive system (PDO), an integrated response of the immune system is formed, mediated by cellular and humoral factors, the ultimate goal of which is to inactivate and remove pathogenic agents from the body. Under physiological conditions, the functioning of the immune system is determined by the balanced production of regulatory cytokines by T-helper types 1 and 11. Violation of the cytokine balance plays a significant role in the chronicity and progression of PDO.

To determine the quantitative content of CK, a highly informative method of enzyme immunoassay with the use of highly sensitive test systems, incl. and domestic production.

The results of long-term studies carried out at the Central Research Institute of Gastroenterology have revealed the peculiarities of changes in the cytokine status in PDO, depending on the etiological factor, course options, stage, duration of the disease, therapy.

For such chronic recurrent diseases of the digestive system (CRID) as peptic ulcer, cholelithiasis, pancreatitis, a multiple, relatively short-term increase in the content of a wide range of CKs in the peripheral blood is characteristic, reflecting the temporal sequence of their synthesis, the dynamics of the pathological process. In the early stages and at the peak of the exacerbation of CRID, in the phase of alterative-destructive processes, an increase in the level of IL-1β, -6, -8, -12, IF-γ, TNF-α prevails (on average - 240-780, reaching in some patients with expressed activity - 1100–3200 pg / ml, in control - up to 40 pg / ml). With an increase in regenerative-restorative processes, the content of pro-inflammatory CK decreases significantly, and anti-inflammatory (IL-4, -10) increases. With the transition to remission in most patients, the concentration of CK approaches normal values. Consequently, in the dynamics of the pathological process in CRDD, the content of CK with various functional properties, their ratio undergoes significant changes.

For such chronic progressive diseases (CPDD), such as chronic hepatitis, cirrhosis of the liver, Crohn's disease, ulcerative colitis, a moderate (on average 160-390 pg / ml), persistent, relatively monotonic increase in the content of key pro- and anti-inflammatory CK is characteristic, which increases with the action of unfavorable factors, the development of complications, concomitant diseases. As the duration of the disease increases, the frequency of recurrences, the synthesis of CK decreases as a result of suppression of the functional activity of the immune system, depletion of its resources, the development of secondary immunodeficiency due to the progression of the disease itself, as well as the inhibitory effect of drug therapy.

Cytokines regulate the intensity of local and systemic pathological processes. Diseases of the stomach, pancreas, gallbladder, liver, small and large intestine are accompanied by changes in the content of CK in the damaged tissue and the adjacent area, which characterizes the intensity of the local immune response. A pronounced increase in the concentration of CK in the peripheral blood is a reflection of the systemic response of the body, in particular the immune, hematopoietic systems, to local organ damage and can serve as one of the indicators of the intensity of the inflammatory and immune processes, activity, and disease progression.

The etiological factor has a significant impact on the level of circulating CK in PDO. Thus, the increase in the content of CK in chronic infectious, inflammatory, autoimmune diseases is more pronounced than in malignant neoplasms, metabolic disorders, hereditary lesions.

An increase in CK synthesis is a secondary phenomenon, the body's response to the action of pathogenic factors. An increase in the concentration of IL-1β, -2, -6, -8, -12, IF-γ, TNF-α in the early stages and at the height of the disease reflects an increase in adhesive, chemotoxic, cytotoxic activity, synthesis of biologically active substances, acute phase proteins, free radicals. These processes cause microcirculation disturbance, development of hyperemia, edema, necrobiosis. In later periods, under the influence of CK (IF-γ, TNF-α, IL-6, -4, -10), damaged cells are phagocytosed, destructive material is utilized, the processes of regeneration, angiogenesis, restoration of the epithelial layer, and growth of fibrous tissue increase. Through the above mechanisms, CKs take part in the pathogenesis of PDO, initiating and regulating exudative-alterative and compensatory-restorative processes in the tissues of the gastrointestinal tract, realizing the interaction between immunocompetent and various specialized cells. Depending on specific conditions, the Central Committee can play the role of both factors of aggression and defense. The protective effect of CK is associated with the activation of innate and acquired immunity by stimulating nonspecific, natural resistance and a specific immune response.

The biological effect of CK under the action of various pathogenic factors (infectious, toxic, mechanical, thermal) is determined by the intensity, duration of antigenic stimulation and is characterized by a lack of specificity. An increase in the synthesis of CK is a universal, nonspecific response of the body to the action of pathogenic agents. Prolonged, intensive synthesis of CK, their excessive release can become a factor in the progression of the pathological process, exerting a direct damaging effect on cells and tissues.

The role of cytokines in the diagnosis of diseases of the digestive system

Changes in the cytokine status in PDOs of different etiology differ in quantitative parameters; however, it is not possible to identify any significant qualitative, specific features. In this regard, it is not possible to speak about the direct diagnostic value of determining the cytokine status, which does not exclude its indirect significance. For example, an increase in the concentration of pro-inflammatory CK in bile indicates the presence of an inflammatory process in the gallbladder. However, the determination of the cytokine status in PDO has an important prognostic value, since the level of pro- and anti-inflammatory CK, their ratio reflects the intensity of alterative-destructive and regenerative-regenerative processes, their dynamics, and disease progression.

Basic therapy in patients with exacerbations of chronic PDO is accompanied in the majority of patients by a significant decrease in elevated serum CK concentrations as compared to the level preceding treatment. These data reflect the positive dynamics of indicators of clinical and laboratory activity of the disease, immune status, the effectiveness of the therapy. The continuing increase in the content of pro-inflammatory CK (primarily TNF-α) against the background of ongoing therapy indicates the absence of pronounced positive changes, the progression of the pathological process.

Cytokine therapy

Achievements of modern molecular biology, biotechnology, immunology, genetics in the study of the structural organization, functional properties of CK serve as the basis for their use for therapeutic purposes in diseases of various organs and systems.

CK can be used as a substitution, stimulating, and inhibitory therapy for the functional activity of the immune system. The therapeutic effect of a number of CKs is due to their ability to enhance the general reactivity of the body, nonspecific protection and specific immunity, to provide antiviral, antibacterial, antitoxic effect. The indication for replacement, compensatory therapy of CK is a decrease in their content, secondary immunodeficiency states, which are often found in chronic progressive infectious, inflammatory, and autoimmune diseases.

Positive results were noted with the use of recombinant preparations of interferons, interleukins that activate local and systemic immunity. Currently, extensive factual material has been obtained regarding the therapeutic effect of recombinant preparations of interferon-α (roferon A, reaferon, intron A), used as a universal non-specific antiviral agent, in particular for viral hepatitis. At the Central Research Institute of Gastroenterology, the use of combined antiviral therapy in patients with chronic viral hepatitis C, including domestic recombinant preparations of interferon-α 2, was accompanied by positive dynamics of indicators of clinical, histological, biochemical, virological activity, and immune status.

A powerful activator of natural resistance is INF-α preparations, inducers of its synthesis (cycloferon, amiksin), which stimulate nonspecific protection, cytotoxic, phagocytic activity, thereby contributing to the destruction and removal of infected, tumor and other defective cells from the body.

In cases of persistent increase in the synthesis of CK in chronic progressive diseases, inhibitors, antagonists of CK are used. These include, in particular, preparations containing monoclonal antibodies to TNFα (infliximab). Intravenous administration of infliximab to patients with ulcerative colitis and Crohn's disease, who were hospitalized at the Central Research Institute of Gastroenterology, was accompanied by a pronounced change in the cytokine status: a decrease in the peripheral blood content of not only TNF-α (from 110 to 55 pg / ml), but also IL-6 (from 60 to 30 pg / ml), with a simultaneous increase in the concentration of IL-12 (from 90 to 210 pg / ml), without a significant change in the level of IL-4.

Thus, the use of CK, their inducers, inhibitors is accompanied by an improvement in the indicators of clinical and laboratory activity, a decrease in the intensity of inflammatory, immunopathological reactions in chronic PDO, however, the positive effect is temporary.

Conclusion

Changes in the cytokine status in PDO are expressed to varying degrees depending on the etiological factor, course options, duration, stage, disease activity, and therapy. The maximum, relatively short-term increase in the content of a wide spectrum of CK in the peripheral blood, reflecting the dynamics of the pathological process, is characteristic of exacerbations of chronic recurrent PDOs. A prolonged, monotonous, moderately pronounced increase in the concentration of key pro- and anti-inflammatory CKs was noted in progressive PDOs. Basic therapy for PDO is accompanied by a decrease in the increased content of CK with a simultaneous positive dynamics of clinical and laboratory indicators of disease activity.

Determination of the cytokine status has an important prognostic value, since it allows one to judge the intensity of inflammatory, infectious, immunopathological processes, their dynamics, the progression of PDO, and the effectiveness of the therapy.

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Cytokines are a special type of protein that can be generated in the body by immune cells and cells in other organs. Most of these cells can be generated by leukocytes.

With the help of cytokines, the body can transfer various information between its cells. Such a substance enters the cell surface and can contact other receptors, transmitting a signal.

These elements are formed and allocated quickly. Different fabrics can participate in their creation. Also, cytokines can have a certain effect on other cells. They can both enhance each other's action and reduce it.

Such a substance can manifest its activity even when its concentration in the body is small. Also, a cytokine can affect the formation of various pathologies in the body. With the help of them, doctors carry out various methods of examining a patient, in particular, in oncology and in infectious diseases.

Cytokine makes it possible to accurately diagnose cancer, and therefore is often used in oncology to establish a residual diagnosis. Such a substance can independently develop and multiply in the body, while not affecting its work. With the help of these elements, any examination of the patient, including in oncology, is facilitated.

They play an important role in the body and have many functions. In general, the work of cytokines is to transfer information from cell to cell and ensure their coordinated work. For example, they can:

• Regulate immune responses.
• Take part in autoimmune reactions.
• Regulate inflammation processes.
• Take part in allergic processes.
• Determine the lifespan of cells.
• Participate in the bloodstream.
• To coordinate the reactions of the body systems when exposed to stimuli.
• Provide a level of toxic effects on the cell.
• Maintain homeostasis.

Doctors have found that cytokines are able to take part not only in the immune process. They also participate in:

1. Normal course of various functions.
2. Fertilization process.
3. Humoral immunity.
4. Recovery processes.

Cytokine classification

Today, scientists know more than two hundred types of these elements. But new species are constantly being discovered. Therefore, to improve the process of understanding this system, doctors have come up with a classification for them. It:

• Regulating inflammatory processes.
• Cells regulating immunity.
• Regulating humoral immunity.

Also, the classification of cytokines determines the presence of certain subspecies in each class. For a more accurate acquaintance with them, you need to look at the information on the network.

Inflammation and cytokines

At the onset of inflammation, the body begins to produce cytokines. They can affect cells that are nearby and transfer information between them. Also, among the cytokines, you can find those that prevent the development of inflammation. They can cause effects that are similar to the manifestation of chronic pathologies.

Pro-inflammatory cytokines

Lymphocytes and tissues can produce such bodies. Cytokines themselves and certain pathogens of infectious diseases can stimulate the production. With a large release of such bodies, local inflammation occurs. With the help of certain receptors, other cells can also be involved in the inflammatory process. All of them also begin to produce cytokines.

The main inflammatory cytokines are TNF-alpha and IL-1. They can adhere to the walls of blood vessels, penetrate into the blood and then spread with it throughout the body. Such elements can synthesize cells that are produced by lymphocytes and affect the foci of inflammation, providing protection.

Also TNF-alpha and IL-1 can stimulate the work of different systems and cause about 40 active other processes in the body. In this case, the effect of cytokines can be exerted on all types of tissues and organs.

Anti-inflammatory cytokines

Anti-inflammatory cytokines can control the above cytokines. They can not only neutralize the effects of the former, but also synthesize proteins.

When inflammation occurs, the amount of these cytokines is important. The complexity of the course of the pathology, its duration and symptoms largely depend on the balance. It is with the help of anti-inflammatory cytokines that blood clotting improves, enzymes are produced and tissue scarring is formed.

Immunity and cytokines

In the immune system, every cell has an important role to play. Through certain reactions, cytokines can control cell interactions. It is they who enable them to exchange important information.

The peculiarity of cytokines is that they have the ability to transmit complex signals between cells and suppress or activate most of the processes in the body. With the help of cytokines, the immune system and others interact.

When the connection is broken, the cells die. This is how complex pathologies appear in the body. The outcome of the disease largely depends on whether the cytokines in the process can establish communication between cells and prevent the introduction of the pathogen into the body.

When the body's defense reaction was not enough to resist the pathology, then cytokines begin to activate other organs and systems that help the body fight infection.

When cytokines exert their effect on the central nervous system, then all human reactions change, hormones and proteins are synthesized. But such changes are not always random. They are either required for protection, or switch the body to combat pathology.

Analyzes

Determining cytokines in the body requires sophisticated testing at the molecular level. With the help of such a test, a specialist can identify polymorphic genes, predict the appearance and course of a particular disease, develop a prophylaxis scheme for ailments, and so on. All this is done purely on an individual basis.

The polymorphic gene can only be found in 10% of the world's population. In such people, an increased activity of immunity can be noted during operations or infectious diseases, as well as other effects on tissues.

When testing such individuals, kipper cells are often identified in the body. Which can cause suppuration after the above procedures or septic disorders. Also, increased activity of immunity in certain cases in life can interfere with a person.

To pass the test, you do not need to prepare specifically for it. For the analysis, you will need to take part of the mucous membrane from the mouth.

Pregnancy

Research has shown that today, pregnant women may have an increased tendency of the body to form blood clots. This can cause termination of pregnancy or infection of the fetus.

When a gene, when carrying a fetus, begins to mutate in the mother's body, then this in 100% of cases becomes the cause of the death of the child. In this case, to prevent the manifestation of this pathology, it will be necessary to first examine the father.

It is these tests that help predict the course of pregnancy and take measures if possible manifestations of certain pathologies. If the risk of pathology is high, then the process of conception may be postponed to another period, during which the father or mother of the unborn child needs to undergo comprehensive treatment.